The dynamin family of proteins consists of unique GTPases involved in membrane fission and fusion events throughout the cell. Our goal is to understand the dynamic structural properties of these proteins and correlate them with their diverse cellular functions. The founding member, dynamin, is crucial for endocytosis, synaptic membrane recycling, membrane trafficking within the cell and, more recently, has been associated with filamentous actin. In the past few years, mutations in dynamin have been shown to cause the peripheral neuropathy, Charcot-Marie-Tooth disease, and centronuclear myopathy (CNM). Dynamin was first implicated in endocytosis when it was discovered to be the mammalian homologue of the shibire gene product in Drosophila. A temperature sensitive shibire allele causes a defect in clathrin-mediated endocytosis. Since then, overexpressing human dynamin mutants in mammalian cells was found to block clathrin-mediated endocytosis. [unreadable] [unreadable] Over the years, our structural work has played a leading role in dissecting the function of dynamin in membrane fission. We have shown that purified dynamin readily assembles into rings and spirals and it forms similar structures on liposomes, generating dynamin-lipid tubes that constrict upon GTP hydrolysis. More recent work, using light microscopy, has confirmed our suggestion that dynamin constricts the underlying lipid bilayer. A potential mechanism for dynamin constriction was revealed when we solved the first three-dimensional structure of dynamin. All evidence supports the hypothesis that dynamin assembles around the necks of clathrin-coated pits where it assists in membrane fission. The tension created by dynamin constricting the neck of coated pits may be sufficient for membrane fission in the cell. The ability of dynamin to constrict and generate a force on the underlying lipid bilayer makes it unique among GTPases as a mechanochemical enzyme. [unreadable] [unreadable] Additional dynamin family members have been implicated in a variety of fundamental cellular processes, including mitochondrial fission and fusion, anti-viral activity, cell plate formation and chloroplast biogenesis. Among these proteins, self-assembly and oligomerization into ordered structures is a common characteristic and, for the majority, is essential for their function. Although there is a wealth of information regarding dynamin, little is known about the structural properties of dynamin-related proteins. We are beginning to address the question of whether a common mechanism of action exists among the dynamin family members. Specifically, we are currently working in collaboration with Dr. Jodi Nunnari (UC Davis) to examine the yeast dynamin-related protein, Dnm1, the master regulator of mitochondrial division. Our work provides clear evidence that dynamin related proteins share common assembly and GTPase properties. However, the structural constraints must still allow for their widely diverse biological functions.[unreadable] [unreadable] In 2001, we solved the first structure of dynamin in the constricted state using helical reconstruction methods. More recently, we solved the structure of dynamin in the non-constricted state using the IHRSR method in collaboration with Dr. Edward Egelman at U. Virginia. The 3D volumes reveal three distinct radial densities, outer, middle and inner layers. During constriction the most obvious change is a decrease in the axial repeat and radius. However, the volume interiors shows a large conformational change within the middle layer, which provides a clue to the mechanism of constriction. Dynamin contains five identifiable domains: GTPase, middle, pleckstrin homology (PH), GTPase effector (GED) and proline/arginine-rich (PRD). Docking of X-ray crystal structures into our maps and biochemical results from other investigators, have revealed that the GTPase domain is located in the outer layer and the PH domain is located in the inner layer. The positioning of GED within the middle layer fits with previous findings that GED directly interacts in trans with a GTPase domain to stimulate the GTPase activity of dynamin. Comparison of the 3D maps in the constricted and non-constricted states shows a large conformational change in the middle layer and suggests GED interacts in trans with the GTPase domain to cause membrane constriction. [unreadable] [unreadable] We have examined the conformational change of dynamin during GTP hydrolysis by time-resolved cryo-electron microscopy to further understand the mechanism of this dynamic process. The method allows the sample to be examined quickly in a thin layer of vitreous ice without the addition of stain or carbon support. We showed that immediately upon GTP addition dynamin constricts the underlying lipid bilayer in a concerted action and excess lipid bulges out at focal points along the constricted tubes. Following constriction, dynamin falls off the lipid bilayer, suggesting that dynamin-dynamin interactions are unstable in the constricted state. Our results demonstrate that dynamin can rapidly constrict the necks of coated pits and disassemble once its role in membrane fission is complete. [unreadable] [unreadable] To determine if a common mechanism of action exists among the dynamin family members, we examined the structure and function of Dnm1, a yeast dynamin family member involved in mitochondria fission. In collaboration with Dr. Jodi Nunnari (UC Davis) we have shown that Dnm1 assembles into large spirals, 100 nm in diameter compared to the 50 nm for dynamin spirals. Remarkably, the diameter of Dnm1 spirals is the same as that of mitochondrial constriction sites observed in cells. Dnm1 also assembles onto liposomes in the absence or presence of nucleotides, forming well-decorated tubes. In addition, the GTP hydrolysis rate of Dnm1 is highly cooperative with respect to its self-assembly state and concentration, which is consistent with the kinetic properties of dynamin. These results suggest that although dynamin family members share common characteristics, their structural properties are uniquely tailored to fit their function. [unreadable] [unreadable] Though Dnm1 can assemble onto liposomes in vitro, their assembly in cells is tightly regulated. We have identified the first protein required for the assembly of a Dnm1 in cells. Two additional mitochondrial proteins, Fis1 and Mdv1, are required and function together with Dnm1 in mitochondrial division. We have shown that Mdv1 interacts with Dnm1 only when Dnm1 is assembled into GTP-bound ring or spiral structures. GTPase mutants defective in binding GTP, which failed to self-assemble into spirals, no longer localized with Mdv1. These findings suggest Mdv1 functions in fission by stabilizing or promoting the formation of Dnm1 into spiral-like structures. Mdv1 may accomplish this by stabilizing the GTP bound form of Dnm1 or by acting as a nucleator, promoting Dnm1 to form spirals at sites of membrane constriction.